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CN-121991245-A - PEDV mRNA vaccine and application thereof

CN121991245ACN 121991245 ACN121991245 ACN 121991245ACN-121991245-A

Abstract

PEDV mRNA vaccine and use thereof. The invention provides mutant PEDV S protein variants, fusion proteins of S protein and chaperones, and fusion proteins of S protein variants and chaperones. Also provided are mRNA vaccines encoding the S protein, the S protein variant, the S protein-chaperone fusion protein, the mRNA of the S protein variant-chaperone fusion protein, and optionally a molecular adjuvant, and uses thereof.

Inventors

  • XIAO XIAO
  • MIAO JIAYING
  • TONG KUN
  • HUANG LEI

Assignees

  • 江苏申基生物科技有限公司

Dates

Publication Date
20260508
Application Date
20241107

Claims (12)

  1. 1. The fusion protein is characterized by comprising Porcine Epidemic Diarrhea Virus (PEDV) S protein or a variant thereof and a molecular chaperone directly or indirectly fused with the Porcine Epidemic Diarrhea Virus (PEDV) S protein, wherein the molecular chaperone is selected from T4 Folden, porcine IgG Fc, ft, recombinant humanized III collagen Rh3C, HIV gp41 6HB, GCN4 or Qalpha; Preferably, the S protein variant is stable in a pre-fusion conformation; Preferably, the variant comprises a mutation selected from one or more of K893P, R894P, A969P, A1032P, D1076P, I1077P, the amino acid position of said mutation being numbered according to the amino acid sequence set forth in any one of the parent SEQ ID NOs 17-20; preferably, the S protein variant has D1076P and I1077P mutations; Preferably, the S protein or S protein variant further comprises a deleted or substituted signal peptide; Preferably, the replacement signal peptide is a replacement of a native signal peptide in the parent with a signal peptide selected from the group consisting of a porcine IL-2 signal peptide, a tPA signal peptide, a porcine IgG heavy chain signal peptide, a porcine IL-6 signal peptide, and a porcine IL-10 signal peptide; Preferably, the replacement signal peptide comprises a signal peptide sequence selected from any one of SEQ ID NOs 46 to 50 or a signal peptide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to any one of SEQ ID NOs 46 to 50; Preferably, the variant comprises the amino acid sequence set forth in any one of SEQ ID NOS 13-16 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to any one of SEQ ID NOS 13-16; preferably, the S protein comprises the amino acid sequence set forth in any one of SEQ ID NOs 17-20 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to any one of SEQ ID NOs 17-20; Preferably, the chaperone is selected from the group consisting of the amino acid sequence set forth in any one of SEQ ID NO:39-45 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to any one of SEQ ID NO: 39-45; Preferably, the fusion protein comprises an amino acid sequence selected from the group consisting of the amino acid sequences set forth in any one of SEQ ID NOs 1-12 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to any one of SEQ ID NOs 1-12; Preferably, the fusion protein comprises the amino acid sequence set forth in any one of SEQ ID NOs 51-58,127-130 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to any one of SEQ ID NOs 51-58, 127-130; Preferably, the fusion protein further fuses an HA tag comprising the amino acid sequence shown as SEQ ID NO: 147.
  2. 2. Nucleic acid encoding the fusion protein of claim 1, preferably comprising a nucleotide sequence selected from the group consisting of the nucleotide sequence set forth in any one of SEQ ID NOS: 59-70 or a degenerate sequence thereof or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to any one of SEQ ID NOS: 59-70, preferably comprising the nucleotide sequence set forth in any one of SEQ ID NOS: 135-146 or a degenerate sequence thereof or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to any one of SEQ ID NOS: 135-146.
  3. 3. Nucleic acid comprising a polynucleotide encoding a Porcine Epidemic Diarrhea Virus (PEDV) S protein or an S protein variant, wherein the S protein variant is stable in a pre-fusion conformation; Preferably, the S protein variant comprises a protein selected from the group consisting of K893P, R894P, A969P, A1032P, D1076P, Mutations in one or more of I1077P; Preferably, the mutated amino acid positions are numbered according to the amino acid sequence shown in any one of the parent SEQ ID NOs 17 to 20; preferably, the S protein variant has D1076P and I1077P mutations; Preferably, the S protein or S protein variant further comprises a deleted or substituted signal peptide; Preferably, the replacement signal peptide is a replacement of a native signal peptide in the parent with a signal peptide selected from the group consisting of a porcine IL-2 signal peptide, a tPA signal peptide, a porcine IgG heavy chain signal peptide, a porcine IL-6 signal peptide, and a porcine IL-10 signal peptide; Preferably, the replacement signal peptide comprises a signal peptide sequence selected from any one of SEQ ID NOs 46 to 50 or a signal peptide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to any one of SEQ ID NOs 46 to 50; Preferably, the nucleic acid encoding the substituted signal peptide is selected from the group consisting of the nucleic acid sequence of any one of SEQ ID NOS: 104-108 or a degenerate sequence thereof or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to any one of SEQ ID NOS: 104-108; Preferably, the subtype of PEDV is selected from GIIa, GIIb, GIIc and GI.
  4. 4. The nucleic acid according to claim 3, wherein, The S protein comprises an amino acid sequence shown in any one of SEQ ID NO 17-20 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with any one of SEQ ID NO 17-20; And/or the nucleic acid encoding the S protein comprises a nucleotide sequence of any one of SEQ ID NOs 75-78 or a degenerate sequence thereof or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to any one of SEQ ID NOs 75-78; And/or the S protein variant comprises the amino acid sequence set forth in any one of SEQ ID NOs 13-16 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to any one of SEQ ID NOs 13-16; And/or the S protein variant encoding nucleic acid comprises a nucleotide sequence of any one of SEQ ID NOS: 71-74 or a degenerate sequence thereof or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to any one of SEQ ID NOS: 71-74.
  5. 5. The nucleic acid of claim 3 or 4, wherein the S protein or S protein variant is fused to a chaperone to form a fusion protein; Preferably, the fusion is a direct fusion or an indirect fusion via a linker; preferably, the linker is selected from the group consisting of a 2A peptide and a GS linker; preferably, the GS linker is selected from (GGGGS) n, (GGGS) n, preferably, n is 2,3,4,5 or 6. Preferably, the chaperone is selected from one or more of T4 Folden (T4 fibritin), porcine IgG Fc, ft, recombinant humanized type III collagen Rh3C, HIV gp41 6HB, GCN4 and qα; Preferably, the chaperone is selected from the group consisting of the amino acid sequence set forth in any one of SEQ ID NO:39-45 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to any one of SEQ ID NO: 39-45; Preferably, the nucleic acid encoding the chaperone comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence set forth in any one of SEQ ID NOS: 97-103 or a degenerate sequence thereof or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to any one of SEQ ID NOS: 97-103; Preferably, the fusion protein comprises an amino acid sequence selected from the group consisting of the amino acid sequences set forth in any one of SEQ ID NOs 1-12 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to any one of SEQ ID NOs 1-12; Preferably, the nucleic acid encoding the fusion protein comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence set forth in any one of SEQ ID NO 59-70 or a degenerate sequence thereof, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to any one of SEQ ID NO 59-70, preferably the fusion protein comprises the amino acid sequence set forth in any one of SEQ ID NO 51-58,127-130 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to any one of SEQ ID NO 51-58,127-130, preferably the nucleic acid encoding the fusion protein comprises the nucleotide sequence set forth in any one of SEQ ID NO 135-146 or a degenerate sequence thereof, or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to any one of SEQ ID NO 135-146; Preferably, the fusion protein further fuses an HA tag comprising the amino acid sequence shown as SEQ ID NO: 147.
  6. 6. The nucleic acid of any one of claims 2-5, which is DNA or RNA; preferably, the nucleic acid is mRNA, circular RNA or self-replicating RNA; preferably, the RNA further comprises a 5'utr, kozak,3' utr, polyA and/or cap structure; preferably, the RNA is a modified RNA in which uracil, cytosine, adenine or guanine nucleotides contain a modifying group; Preferably, wherein the modifying group is selected from at least one of pseudouridine, N1-methyl pseudouridine, N1-ethyl pseudouridine, 5-methylcytosine, 5-methoxycytosine, N1-methylcytosine, 2-thiouridine, 5-methoxyuridine or N1-methyladenosine, N1-methylguanine, isoguanine; Preferably, wherein the mRNA molecule is a modified mRNA, the modification of which includes conversion of uridine to pseudouridine, N1-methyl pseudouridine, N1-ethyl pseudouridine, 2-thiouridine, 5-methoxyuridine, and/or conversion of cytosine nucleoside to 5-methylcytosine, 5-methoxycytosine, N1-methylcytosine, and/or conversion of adenine nucleoside to N1-methyladenosine, and/or conversion of adenine nucleoside to N1-methylguanine, isoguanine.
  7. 7. The nucleic acid of claim 6, which is mRNA; preferably, the 5' UTR comprises the nucleotide sequence of any one of SEQ ID NOs 109,111,113,115 or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to any one of SEQ ID NOs 109,111,113, 115; Preferably, the 3' UTR comprises the nucleotide sequence shown in any one of SEQ ID NOs 110,112,114,116 or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to any one of SEQ ID NOs 110,112,114, 116; Preferably, the polyA comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 117-118 or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to any one of SEQ ID NOS: 117-118; Preferably, the nucleic acid comprises a nucleotide sequence as set forth in any one of SEQ ID NOS 119-126,131-134 or a degenerate sequence or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to any one of SEQ ID NOS 119-126,131-134; Preferably, the cap structure is a compound of formula (I), or a pharmaceutically acceptable salt, stereoisomer, tautomer, or isotopic variant thereof: Wherein, the Is a single bond or is not present, X 1 is selected from O、S、CH 2 、CH 2 CH 2 、CH＝CH、CH＝CHO、CH 2 O、OCH 2 、CH 2 CH 2 O、 OCH 2 CH 2 , ternary cycloalkyl, R 1 、R 2 、R 3 and R 4 are each independently halogen, OH, O-C 1-3 alkyl which is unsubstituted or substituted by O-C 1-3 alkyl, O-C 1-3 alkyl which is unsubstituted or substituted by O-C 1-3 alkyl, B 1 and B 2 are each independently selected from natural, modified or unnatural nucleobases, Preferably, the compound of formula (I) is any one of the following:
  8. 8. Nucleic acid composition, characterized in that it comprises a nucleic acid according to any one of claims 2 to 7 and a molecular adjuvant, preferably selected from the group consisting of TGF-β-P2A-BAFF(SMA-6)、CD40L-P2a-aPRIL、IL-10-P2A-IL-2、GM-CSF-P2A-IL-2、GM-CSF-P2A-CD40L、GM-CSF-P2A-IL-18、IL-17-P2A-IL-21(SMA-7)、IL-17、IL-21、IL-10、IL-2、GM-CSF、IL-18、CCL28、CD40L、BAFF、APRIL、TGF-β; Preferably, the molecular adjuvant is of porcine origin; Preferably, the molecular adjuvant comprises the amino acid sequence of any one of SEQ ID NOs 21-38 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to any one of SEQ ID NOs 21-38; preferably, the nucleic acid encoding the molecular adjuvant comprises a nucleotide sequence set forth in any one of SEQ ID NOS: 79-96 or a degenerate sequence thereof or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to any one of SEQ ID NOS: 79-96; preferably, the composition is a vaccine composition.
  9. 9. A vector comprising the nucleic acid of any one of claims 2-7 or the nucleic acid composition of claim 8, preferably the vector is an expression vector.
  10. 10. A cell comprising the vector of claim 9, preferably the cell is a eukaryotic cell or a prokaryotic cell.
  11. 11. A pharmaceutical composition comprising the fusion protein of claim 1 or the nucleic acid of any one of claims 2-7 or the nucleic acid composition of claim 8 or the vector of claim 9 or the cell of claim 10, preferably the delivery vehicle of the pharmaceutical composition comprises Lipid Nanoparticles (LNP), viral vectors such as AAV, polymeric materials such as Polyethylenimine (PEI), polyamino esters (PBAE), chitosan or polypeptides such as cell penetrating peptides; Preferably, the Lipid Nanoparticle (LNP) comprises an ionizable lipid, DSPC, cholesterol, and DMG-PEG2000 ethanol; preferably, the Lipid Nanoparticle (LNP) is a targeted LNP (tLNP); Preferably, the pharmaceutical composition is formulated in a form suitable for intramuscular or subcutaneous injection, oral administration or inhalation.
  12. 12. Use of the fusion protein of claim 1 or the nucleic acid of any one of claims 2-7 or the nucleic acid composition of claim 8 or the vector of claim 9 or the cell of claim 10 or the pharmaceutical composition of claim 11 for the manufacture of a medicament for the prevention or treatment of Porcine Epidemic Diarrhea Virus (PEDV) infection or a disease caused by PEDV infection, preferably the medicament is a vaccine, preferably the disease is porcine epidemic diarrhea.

Description

PEDV mRNA vaccine and application thereof Technical Field The invention relates to the technical field of biopharmaceuticals, in particular to a vaccine for preventing PEDV mRNA and application thereof. Background The pathogen of Porcine epidemic diarrhea (Porcine EPIDEMIC DIARRHEA, PED) is PEDV, the Porcine epidemic diarrhea has high infectivity, and the PEDV infection can cause clinical symptoms such as vomiting, diarrhea, dehydration and the like, so that piglets are most susceptible, the morbidity of the Porcine epidemic diarrhea is 100% in 1-7 days, and the mortality is 75% -100%. Genetic analysis of the recently developed PEDV strain sequences has found that the current PEDV variant strain GII population is more than 90% and is the main epidemic strain, including GII-a population and GII-b population, wherein the GII-a population is increased year by year, and the S-INDEL strain is present. The virulence and harm of strains of different sub-groups of PEDV are different, the virulence of the variant strain is strong, the death rate of piglets below 7 days of infection is up to 100%, the method is mainly characterized by congestion and thinning of intestinal walls of small intestines, severe diarrhea and dehydration of pigs, the classical strain has weaker virulence, the intestinal villus lesions of the infected pigs are lighter, the diarrhea degree is slight, and the S-INDEL strain has the characteristics of the same morbidity, reduced pathological injury, reduced diarrhea, reduced death rate and the like compared with the GII strain. The existing PEDV vaccines comprise inactivated vaccines, subunit vaccines, attenuated live vaccines, DNA nucleic acid vaccines, vector vaccines and the like. The first attenuated inactivated vaccine based on PEDV GII group variant strain (AJ 1102 strain) is marketed in China, but the vaccine has a remarkably reduced protective effect along with continuous variation of PEDV. The subunit vaccine of PEDV has a good humoral immune response, but cannot stimulate obvious cellular immunity, thus causing a major obstacle to development of the subunit vaccine of PEDV. In 2015, china also marketed diarrhea triple (PEDV, TGEV and PoRV) attenuated vaccines. However, attenuated vaccines have the disadvantages of high transportation and storage costs, strong toxicity, risk of reversion, and the like. DNA nucleic acid vaccines are usually directly injected in the form of recombinant plasmids, which can stably express antigens in host cells for a long time, but can also integrate into the host cell genome for a long time, and thus the safety of DNA vaccines is yet to be further verified. The virus vector vaccine is prepared by inserting PEDV S genes into a vector through genetic engineering biotechnology, expressing antigen proteins by virtue of the virus vector, and can be effectively and safely applied to pigs at present, because the virus vector may have strong side reactions such as red swelling, hot pain, allergy and the like at injection sites, and the recombinant vector vaccine still has the possibility of genetic recombination with other corresponding viruses in the body. The mRNA vaccine is the third generation vaccine after the inactivated vaccine, the attenuated live vaccine, the subunit vaccine and the viral vector vaccine. Compared with the traditional vaccine, the vaccine has good immune effect, is far superior to the traditional vaccine in research and development and production cycle, and has been effectively applied to a plurality of infectious diseases. Disclosure of Invention The present invention is based, in part, on the discovery by the inventors that mRNA encoding type PEDVI, type GIIa, type GIIb, and type GIIc S proteins, S protein variants, S protein-chaperone fusion proteins, S protein variant-chaperone fusion proteins, and optionally molecular adjuvants, can elicit the production and cellular immune responses of human milk, binding antibodies in mice, neutralizing antibodies. The first aspect of the present invention provides a fusion protein comprising a Porcine Epidemic Diarrhea Virus (PEDV) S protein or variant thereof, and a chaperone fused directly or indirectly thereto, selected from T4 Folden, porcine IgG Fc, ft, recombinant humanized type III collagen Rh3C, HIV gp41 HB, GCN4 or qα. In one embodiment, the S protein variant is stable in a pre-fusion conformation. In one embodiment, the variant comprises a mutation selected from one or more of K893P, R894P, A969P, A1032P, D1076P, I1077P, the amino acid position of said mutation being numbered according to the amino acid sequence set forth in any one of the parent SEQ ID NOS: 17-20. In one embodiment, the S protein variant has D1076P and I1077P mutations. In one embodiment, the S protein or S protein variant further comprises a deleted or substituted signal peptide. In one embodiment, the replacement signal peptide is a replacement of a native signal peptide in the parent with a signal peptide selected fro